The long-term objective of this application is to evaluate a specific molecular target for discovery and clinical testing of new anticancer agents, based on molecular mechanisms that underlie chemotherapy resistance. Current research in anticancer drug mechanism of action and resistance biology has identified ceramide glycosylation as an event associated with progression of cancer. The focus of this application is to target glycosylation because it is essential for maintenance of the drug-resistant phenotype. It is believed that a new genre of agents can be brought forth for clinical assessment based on strong rationales. Drug-resistant human cancer cells which are known to demonstrate enhanced metabolism of ceramide through the glycosylation pathway catalyzed by glucosylceramide synthase (GCS), will be exposed to conventional chemotherapy, Adriamycin, Taxol, N-4(hydroxyphenyl)retinamide(4-HPR) in the absence or presence of agents that retard ceramide glycosylation, the latter being evaluated through screening of compounds in the Natural Products and Synthetic Repositories of the NCI. We will conduct analog searches of the repositories using lead compounds already shown to retard ceramide glycosylation and demonstrate synergistic cytotoxicity with classical chemotherapeutic agents. Other programs such as COMPARE and clustering algorithms will be employed to mine the NCI drug repositories. The impact of select agents on ceramide glycosylation will be evaluated in intact cancer cells and in in vitro cell-free assays, and based on results, regimens will be assessed for cytotoxicity either alone or in combination with chemotherapy, using standard cell proliferation and apoptosis assays. This study has two specific aims: 1) to screen compounds in the NCI Natural and Synthetic Products Repositories for ability to block ceramide metabolism by the glycosylation route; 2) to evaluate lead compounds alone and in combination with anticancer drugs for enhancement of chemotherapy-induced cytotoxicity in drug-resistant cancer cells. The limited efficacy of available drug therapies and the high incidence of chemotherapy resistance are strong reasons to pursue new approaches to treat patients with cancer. Dysfunctional ceramide metabolism is a significant contributor to chemo- and radiotherapy failure. Targeting ceramide metabolism is therefore an attractive strategy for anticancer drug development.